Hydrophilic Reduction-Resistant Spin Labels of Pyrrolidine and Pyrroline Series from 3,4-Bis-hydroxymethyl-2,2,5,5-tetraethylpyrrolidine-1-oxyl

Highly resistant to reduction nitroxides open new opportunities for structural studies of biological macromolecules in their native environment inside living cells and for functional imaging of pH and thiols, enzymatic activity and redox status in living animals. 3,4-Disubstituted nitroxides of 2,2,5,5-tetraethylpyrrolidine and pyrroline series with a functional group for binding to biomolecules and a polar moiety for higher solubility in water and for more rigid attachment via additional coordination to polar sites were designed and synthesized. The EPR spectra, lipophilicities, kinetics of the reduction in ascorbate-containing systems and the decay rates in liver homogenates were measured. The EPR spectra of all 3,4-disubstituted pyrrolidine nitroxides showed additional large splitting on methylene hydrogens of the ethyl groups, while the spectra of similar pyrroline nitroxides were represented with a simple triplet with narrow lines and hyperfine structure of the nitrogen manifolds resolved in oxygen-free conditions. Both pyrrolidine and pyrroline nitroxides demonstrated low rates of reduction with ascorbate, pyrrolidines being a bit more stable than similar pyrrolines. The decay of positively charged nitroxides in the rat liver homogenate was faster than that of neutral and negatively charged radicals, with lipophilicity, rate of reduction with ascorbate and the ring type playing minor role. The EPR spectra of N,N-dimethyl-3,4-bis-(aminomethyl)-2,2,5,5-tetraethylpyrrolidine-1-oxyl showed dependence on pH with pKa = 3, ΔaN = 0.055 mT and ΔaH = 0.075 mT.


Introduction
Nitroxides (aminoxyl radicals) are widely used as molecular probes and labels for the investigation of complex molecular systems using various methods of EPR and NMR spectroscopy and imaging [1][2][3][4][5][6][7][8][9][10].Biophysics and structural biology are the areas where application of nitroxide-based spectroscopic methods have been especially fruitful.Special approaches and techniques have been developed for structural studies of biological macromolecules in their native environment inside living cells [2,11] and for functional imaging of pH and thiols [2,12], enzyme activity [13] and redox status [14] in living animals.The stability of the nitroxide radical is of crucial importance for these applications.Early studies showed that reduction of a nitroxide group to hydroxylamine one with enzymatic systems and low-molecular biogenic antioxidants is a major pathway of nitroxide decay in biological systems [15].To slow down a radical decay, so-called "sterically shielded" pyrroline and pyrrolidine nitroxides with four ethyl (or more bulky alkyl) substituents adjacent to the N-O group were designed [16,17].These nitroxides showed much higher stability in biological samples and in cells than conventional tetramethyl-substituted nitroxides [18][19][20][21].A number of reduction-resistant spin labels for in-cell applications have been prepared from these nitroxides [20][21][22].However, bulky alkyl substituents make these spin labels lipophilic and poorly soluble in water.Stock solutions were usually prepared in DMSO or in water-DMSO mixtures, see for example [20,23].Recent studies revealed that an alternative metabolic pathway implying cytochrome P450 activity may significantly contribute to the decay of sterically shielded nitroxides in subcellular systems [24,25].These data coincide with our observations that decay of 2,2,5,5-tetraethylpyrrolidine nitroxides in homogenates of liver and kidney, which are known to be rich of cytochrome P450 enzymes, proceeds much faster than in homogenates of brain, heart or in blood [22].According to some estimates, hydrophilic nitroxides, with a negative charge and/or minimal hydrophilicity are required to slow down transformation by P450 enzymes, either via reductive or oxidative metabolism [24].
Early studies showed that reduction of a nitroxide group to hydroxylamine one with enzymatic systems and low-molecular biogenic antioxidants is a major pathway of nitroxide decay in biological systems [15].To slow down a radical decay, so-called "sterically shielded" pyrroline and pyrrolidine nitroxides with four ethyl (or more bulky alkyl) substituents adjacent to the N-O group were designed [16,17].These nitroxides showed much higher stability in biological samples and in cells than conventional tetramethyl-substituted nitroxides [18][19][20][21].A number of reduction-resistant spin labels for in-cell applications have been prepared from these nitroxides [20][21][22].However, bulky alkyl substituents make these spin labels lipophilic and poorly soluble in water.Stock solutions were usually prepared in DMSO or in water-DMSO mixtures, see for example [20,23].Recent studies revealed that an alternative metabolic pathway implying cytochrome P450 activity may significantly contribute to the decay of sterically shielded nitroxides in subcellular systems [24,25].These data coincide with our observations that decay of 2,2,5,5-tetraethylpyrrolidine nitroxides in homogenates of liver and kidney, which are known to be rich of cytochrome P450 enzymes, proceeds much faster than in homogenates of brain, heart or in blood [22].According to some estimates, hydrophilic nitroxides, with a negative charge and/or minimal hydrophilicity are required to slow down transformation by P450 enzymes, either via reductive or oxidative metabolism [24].
Scheme 1.General approach to the synthesis of hydrophilic reduction-resistant spin labels.
The aim of this work was to find a convenient synthetic approach to 2,2,5,5-tetraethyl-3,4-disubstituted pyrrolidine and pyrroline nitroxides with two different functional groups: one for selective binding to biopolymers or other target molecules (X) and a polar charged moiety that makes the nitroxide hydrophilic and water soluble (Y).We have focused on the preparation of hydrophilic analogs of the known reduction-resistant maleimide spin labels, which are used for selective binding to thiol groups of cysteine residues [20,21], and nitroxide azides, which can be used inside the cells for bioorthogonal spin Scheme 1.General approach to the synthesis of hydrophilic reduction-resistant spin labels.
The aim of this work was to find a convenient synthetic approach to 2,2,5,5-tetraethyl-3,4-disubstituted pyrrolidine and pyrroline nitroxides with two different functional groups: one for selective binding to biopolymers or other target molecules (X) and a polar charged moiety that makes the nitroxide hydrophilic and water soluble (Y).We have focused on the preparation of hydrophilic analogs of the known reduction-resistant maleimide spin labels, which are used for selective binding to thiol groups of cysteine residues [20,21], and nitroxide azides, which can be used inside the cells for bioorthogonal spin labeling of proteins modified with unnatural alkyne-containing amino acids using copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) [36] or strain-promoted azide-alkyne cycloadditions (SPAAC) [37].Since 1 is a readily available compound, novel hydrophilic nitroxides can be produced on a large scale not only for SDSL-EPR studies but also for the development of specific probes/contrast agents for in vivo EPRI/OMRI/MRI studies.The parameters of EPR spectra of the new nitroxides, their lipophilicities and decay in liver homogenate and in model ascorbate-containing systems were studied.

Synthesis of the Hydrophilic Nitroxides
To convert a symmetric structure, such as 1, into heterofunctional spin labels one should find a convenient way to modify only one of the two identical functional groups with a satisfactory yield.Reactions on one of two hydroxyl groups in 1 seems to be the shortest pathway to heterobifunctional derivatives of 2,2,5,5-tetraethylpyrrolidine-1-oxyl.The subsequent selective modification of the substituents in the position 3 and 4 can be used to prepare various derivatives.We have showed earlier that the reaction of 1 with excess of methanesulfonyl chloride (MsCl) in the presence of diisopropylethylamine (DIPEA) afforded 3 (Scheme 2) [28], and the latter was used in the synthesis of 2. Notably, we did not observe any significant conversion in the absence of a base, while the reaction with MsCl in the presence of excess of the base caused accumulation of dark-colored products.Therefore, we used slow addition of DIPEA to control the reaction in the presence of excess of MsCl.The addition of a smaller amount of reagents gives a mixture of di-and monomesylated products 3 and 4. The optimal preparative yield of 4 64-68% (75-80% with conversion ca.85%) was achieved upon slow addition of DIPEA (1.4 eq.) to a solution of 1 and MsCl (1.58 eq.).The higher excess of the reagents required for nearly quantitative conversion of 1 with yields of 3 and 4 are 35 and 55%, correspondingly.Both 3 and 4 were isolated as crystalline compounds.Structure assignments were done on the basis of NMR spectra after nitroxide reduction with Zn/CF 3 COOH [22], IR spectra, element analyses (see Section 3 and Supplementary Materials pp.S4, S10) and single crystal X-ray data (Figure 1).An unexpectedly high yield of 4 may result from stabilization of anionic form of 1 via hydrogen bonding with the neighboring hydroxy group, which makes the rate of mesylation of 1 much higher than that of 4.
labeling of proteins modified with unnatural alkyne-containing amino acids using copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) [36] or strain-promoted azide-alkyne cycloadditions (SPAAC) [37].Since 1 is a readily available compound, novel hydrophilic nitroxides can be produced on a large scale not only for SDSL-EPR studies but also for the development of specific probes/contrast agents for in vivo EPRI/OMRI/MRI studies.The parameters of EPR spectra of the new nitroxides, their lipophilicities and decay in liver homogenate and in model ascorbate-containing systems were studied.

Synthesis of the Hydrophilic Nitroxides
To convert a symmetric structure, such as 1, into heterofunctional spin labels one should find a convenient way to modify only one of the two identical functional groups with a satisfactory yield.Reactions on one of two hydroxyl groups in 1 seems to be the shortest pathway to heterobifunctional derivatives of 2,2,5,5-tetraethylpyrrolidine-1-oxyl.The subsequent selective modification of the substituents in the position 3 and 4 can be used to prepare various derivatives.We have showed earlier that the reaction of 1 with excess of methanesulfonyl chloride (MsCl) in the presence of diisopropylethylamine (DI-PEA) afforded 3 (Scheme 2) [28], and the latter was used in the synthesis of 2. Notably, we did not observe any significant conversion in the absence of a base, while the reaction with MsCl in the presence of excess of the base caused accumulation of dark-colored products.Therefore, we used slow addition of DIPEA to control the reaction in the presence of excess of MsCl.The addition of a smaller amount of reagents gives a mixture of di-and monomesylated products 3 and 4. The optimal preparative yield of 4 64-68% (75-80% with conversion ca.85%) was achieved upon slow addition of DIPEA (1.4 eq.) to a solution of 1 and MsCl (1.58 eq.).The higher excess of the reagents required for nearly quantitative conversion of 1 with yields of 3 and 4 are 35 and 55%, correspondingly.Both 3 and 4 were isolated as crystalline compounds.Structure assignments were done on the basis of NMR spectra after nitroxide reduction with Zn/CF3COOH [22], IR spectra, element analyses (see Section 3 and Supplementary Materials pp.S4, S10) and single crystal X-ray data (Figure 1).An unexpectedly high yield of 4 may result from stabilization of anionic form of 1 via hydrogen bonding with the neighboring hydroxy group, which makes the rate of mesylation of 1 much higher than that of 4.   Int.J. Mol.Sci.2024, 25, x FOR PEER REVIEW labeling of proteins modified with unnatural alkyne-containing amino acids using c (I)-catalyzed azide-alkyne cycloaddition (CuAAC) [36] or strain-promoted azide-a cycloadditions (SPAAC) [37].Since 1 is a readily available compound, novel hydro nitroxides can be produced on a large scale not only for SDSL-EPR studies but also f development of specific probes/contrast agents for in vivo EPRI/OMRI/MRI studie parameters of EPR spectra of the new nitroxides, their lipophilicities and decay in homogenate and in model ascorbate-containing systems were studied.

Synthesis of the Hydrophilic Nitroxides
To convert a symmetric structure, such as 1, into heterofunctional spin labe should find a convenient way to modify only one of the two identical functional g with a satisfactory yield.Reactions on one of two hydroxyl groups in 1 seems to shortest pathway to heterobifunctional derivatives of 2,2,5,5-tetraethylpyrrolidine-1 The subsequent selective modification of the substituents in the position 3 and 4 c used to prepare various derivatives.We have showed earlier that the reaction of 1 excess of methanesulfonyl chloride (MsCl) in the presence of diisopropylethylamin PEA) afforded 3 (Scheme 2) [28], and the latter was used in the synthesis of 2. Notab did not observe any significant conversion in the absence of a base, while the reaction MsCl in the presence of excess of the base caused accumulation of dark-colored pro Therefore, we used slow addition of DIPEA to control the reaction in the presence cess of MsCl.The addition of a smaller amount of reagents gives a mixture of d monomesylated products 3 and 4. The optimal preparative yield of 4 64-68% (75 with conversion ca.85%) was achieved upon slow addition of DIPEA (1.4 eq.) to a so of 1 and MsCl (1.58 eq.).The higher excess of the reagents required for nearly quanti conversion of 1 with yields of 3 and 4 are 35 and 55%, correspondingly.Both 3 and 4 isolated as crystalline compounds.Structure assignments were done on the basis of spectra after nitroxide reduction with Zn/CF3COOH [22], IR spectra, element analyse Section 3 and Supplementary Materials pp.S4, S10) and single crystal X-ray data (F 1).An unexpectedly high yield of 4 may result from stabilization of anionic form o hydrogen bonding with the neighboring hydroxy group, which makes the rate of m ation of 1 much higher than that of 4.   Mesyloxy derivatives are convenient precursors for a variety of spin labels.Heating of 4 with an excess of sodium azide in DMSO afforded 5 with nearly quantitative yield (Scheme 3).Azido group was then reduced with LiAlH 4 to give aminoalcohol 6 which was isolated as crystalline solid.The structure of 6 was confirmed by IR and NMR spectra and single crystal X-ray analyses data (Figure 1 and Supplementary Materials pp.S5, S12).
Mesyloxy derivatives are convenient precursors for a variety of spin labels.Heating of 4 with an excess of sodium azide in DMSO afforded 5 with nearly quantitative yield (Scheme 3).Azido group was then reduced with LiAlH4 to give aminoalcohol 6 which was isolated as crystalline solid.The structure of 6 was confirmed by IR and NMR spectra and single crystal X-ray analyses data (Figure 1 and Supplementary Materials pp.S5, S12).

Scheme 3. Synthesis of 6.
A reaction with N-methoxycarbonyl maleimide (NMCM) was chosen to convert amine 6 into corresponding spin label (Scheme 4).The use of this reagent made it possible to keep the hydroxy group intact, although the yield of spin label 7 was low.In this and all subsequent similar syntheses of maleimide spin labels, we used a modified protocol based on literature methods [38,39].Primary amine was slowly added dropwise to a solution of NMCM in dry THF at 0 °C under argon, and then the reaction mixture was diluted with a saturated aqueous solution of NaHCO3.This two-step procedure prevents the addition of unreacted amine to the maleimide formed, since the elimination of methylcarbamate to form maleimide occurs in the second stage, when the amine has already been consumed.Despite the presence of a hydrophilic hydroxy group, radicals 5 and 7 are still poorly soluble in water.The introduction of quaternary ammonium groups, such as trimethylammonium, is a simple and effective way to make a compound highly hydrophilic.It is more convenient to introduce such groups at the final steps of synthesis.For this reason, amino group in 6 was protected with tert-butyloxycarbonyl moiety to give 8.The latter was carefully treated with methanesulfonyl chloride in basic conditions (excess of DIPEA) Scheme 3. Synthesis of 6.
A reaction with N-methoxycarbonyl maleimide (NMCM) was chosen to convert amine 6 into corresponding spin label (Scheme 4).The use of this reagent made it possible to keep the hydroxy group intact, although the yield of spin label 7 was low.In this and all subsequent similar syntheses of maleimide spin labels, we used a modified protocol based on literature methods [38,39].Primary amine was slowly added dropwise to a solution of NMCM in dry THF at 0 • C under argon, and then the reaction mixture was diluted with a saturated aqueous solution of NaHCO 3 .This two-step procedure prevents the addition of unreacted amine to the maleimide formed, since the elimination of methylcarbamate to form maleimide occurs in the second stage, when the amine has already been consumed.
Mesyloxy derivatives are convenient precursors for a variety of spin labels.Heating of 4 with an excess of sodium azide in DMSO afforded 5 with nearly quantitative yield (Scheme 3).Azido group was then reduced with LiAlH4 to give aminoalcohol 6 which was isolated as crystalline solid.The structure of 6 was confirmed by IR and NMR spectra and single crystal X-ray analyses data (Figure 1 and Supplementary Materials pp.S5, S12).

Scheme 3. Synthesis of 6.
A reaction with N-methoxycarbonyl maleimide (NMCM) was chosen to convert amine 6 into corresponding spin label (Scheme 4).The use of this reagent made it possible to keep the hydroxy group intact, although the yield of spin label 7 was low.In this and all subsequent similar syntheses of maleimide spin labels, we used a modified protocol based on literature methods [38,39].Primary amine was slowly added dropwise to a solution of NMCM in dry THF at 0 °C under argon, and then the reaction mixture was diluted with a saturated aqueous solution of NaHCO3.This two-step procedure prevents the addition of unreacted amine to the maleimide formed, since the elimination of methylcarbamate to form maleimide occurs in the second stage, when the amine has already been consumed.Despite the presence of a hydrophilic hydroxy group, radicals 5 and 7 are still poorly soluble in water.The introduction of quaternary ammonium groups, such as trimethylammonium, is a simple and effective way to make a compound highly hydrophilic.It is more convenient to introduce such groups at the final steps of synthesis.For this reason, amino group in 6 was protected with tert-butyloxycarbonyl moiety to give 8.The latter was carefully treated with methanesulfonyl chloride in basic conditions (excess of DIPEA) Despite the presence of a hydrophilic hydroxy group, radicals 5 and 7 are still poorly soluble in water.The introduction of quaternary ammonium groups, such as trimethylammonium, is a simple and effective way to make a compound highly hydrophilic.It is more convenient to introduce such groups at the final steps of synthesis.For this reason, amino group in 6 was protected with tert-butyloxycarbonyl moiety to give 8.The latter was carefully treated with methanesulfonyl chloride in basic conditions (excess of DIPEA) to give 9 and subsequent heating with NaN 3 according to the procedure described above afforded 10.The structures of 8 and 10 were assigned on the basis of IR spectra and HRMS (see Section 3 and Supplementary Materials pp.S11-S12) and confirmed with single-crystal X-ray analysis data (Figure 2).Under Eschweiler-Clarke reaction conditions deprotection and alkylation occur with formation of 11a as a single product.
to give 9 and subsequent heating with NaN3 according to the procedure described above afforded 10.The structures of 8 and 10 were assigned on the basis of IR spectra and HRMS (see Section 3 and Supplementary Materials pp.S11-S12) and confirmed with single-crystal X-ray analysis data (Figure 2).Under Eschweiler-Clarke reaction conditions deprotection and alkylation occur with formation of 11a as a single product.We have found that 11a can also be prepared from 3 (Scheme 5) with a higher yield and in fewer steps (45% overall yield from 1 in 4 steps versus 35-36% in 7 steps via Schemes 1-4).It is noteworthy that a similar radical of the pyrroline series, 11b, can also be obtained from 2 using this scheme.It is difficult to obtain a good yield of the monoazido derivative by treating 2 or 3 with sodium azide, see for example literature data for similar 2,2,5,5-tetramethyl-substituted nitroxide [40].In contrast, the corresponding diazides 12a,b were readily obtained by heating of 2 and 3 with excess of NaN3 in DMSO.These nitroxides were isolated as orange crystals with 93-99% yield and characterized with IR spectra, HRMS and element analyses (see Section 3 and Supplementary Materials pp.S11-S12).The structure of 12a was confirmed by single crystal X-ray data (Figure 2).We have found that 11a can also be prepared from 3 (Scheme 5) with a higher yield and in fewer steps (45% overall yield from 1 in 4 steps versus 35-36% in 7 steps via Schemes 1-4).It is noteworthy that a similar radical of the pyrroline series, 11b, can also be obtained from 2 using this scheme.It is difficult to obtain a good yield of the monoazido derivative by treating 2 or 3 with sodium azide, see for example literature data for similar 2,2,5,5tetramethyl-substituted nitroxide [40].In contrast, the corresponding diazides 12a,b were readily obtained by heating of 2 and 3 with excess of NaN 3 in DMSO.These nitroxides were isolated as orange crystals with 93-99% yield and characterized with IR spectra, HRMS and element analyses (see Section 3 and Supplementary Materials pp.S11-S12).The structure of 12a was confirmed by single crystal X-ray data (Figure 2).For the preparation of maleimide spin labels from 11a,b we used the Staudinger reaction followed by treatment with NMCM in analogy to the above procedures.The structures of 15a,b were confirmed by single crystal X-ray analysis (Figure 3).For the preparation of maleimide spin labels from 11a,b we used the Staudinger reaction followed by treatment with NMCM in analogy to the above procedures.The structures of 15a,b were confirmed by single crystal X-ray analysis (Figure 3).For the preparation of maleimide spin labels from 11a,b we used the Staudinger reaction followed by treatment with NMCM in analogy to the above procedures.The structures of 15a,b were confirmed by single crystal X-ray analysis (Figure 3).Diazide 12a can also be converted into dianionic spin label.A Huisgen cycloaddition reaction with one equivalent of dimethyl acetylenedicarboxylate (DMAD) gave a mixture of mono-and bis-triazoles 18 and 19 (Scheme 6).After alkaline hydrolysis of the ester Diazide 12a can also be converted into dianionic spin label.A Huisgen cycloaddition reaction with one equivalent of dimethyl acetylenedicarboxylate (DMAD) gave a mixture of mono-and bis-triazoles 18 and 19 (Scheme 6).After alkaline hydrolysis of the ester groups in 18, the monosodium salt 20 was isolated.The structure of this nitroxide was confirmed by single-crystal X-ray diffraction data (Figure 3).groups in 18, the monosodium salt 20 was isolated.The structure of this nitroxide was confirmed by single-crystal X-ray diffraction data (Figure 3).Scheme 6. Synthesis of hydrophilic spin label 20 using Huisgen 1,3-dipolar cycloaddition reaction.
Table 1.Parameters of the EPR spectra of nitroxides in 50 mM phosphate buffer (pH 7.4) with the addition of 1% DMSO at concentration of 0.2 mM (a N -hfc constant on nitrogen , a H -hfc constant on methylene hydrogens of ethyl groups, H p-p -peak to peak linewidth), partition coefficients (K p ) in octanol/aqueous 5 mM phosphate-citrate-borate buffer pH 7.4 mixtures, and rates of reduction with ascorbate (k red ) (100-300 mM) in 5 mM phosphate-citrate-borate buffer, pH 7.4, 25   The EPR spectra of 3,4-disubstituted pyrrolidine nitroxides correspond to the pattern described for 1, with broad lines and two large additional doublet splittings on two hydrogen atoms of the methylene groups of the ethyl substituents [26,41] (Figure 5a).The value of these constants depends on the size of the neighboring substituent at position 3 or 4. For example, the introduction of the bulky maleimido group results in up to a 50% increase in one of two aH (cf.14a and 15a; 16a and 17a).We have showed earlier that high hfc with one of methylene hydrogens of ethyl group is observed in nitroxides with the pseudoaxial position of this ethyl group and CH2-CH3 bond nearly parallel to the N-O bond axis [41].Apparently, the repulsion of the large substituent and the neighboring ethyl group (cis relative to each other) increases the contribution of this conformation.In contrast, EPR spectra of the pyrroline nitroxides showed classical triplet with no additional large splittings (Figure 5b).In oxygen-free solution, hyperfine structure of the spectral lines was resolved, which is typical for the nitroxides with planar heterocycle (pyrroline, 3-imidazoline and isoindoline) (Figure 5d) [4].The simulation gives four aH = 0.0611 mT and four aH = 0.0317 mT, apparently due to hfc with methylene hydrogens of ethyl groups.
The EPR spectra of nitroxides with ionizable groups may be dependent on pH.Similarly to that described for 3-aminomethyl-proxyl (25) [42] (Figure 4), spectra of 3-aminomethyl-and 3-dimethylaminomethyl nitroxides 6, 11a,b, 13a,b and 15a,b demonstrate small dependence on pH with ΔaN ca.0.02 mT and pK ca. 9. Titration of the nitroxides 14a and 14b revealed two transitions at pH ca. 3 and ca.11 (Figure 6), which apparently correspond to two protonation steps with formation of mono-and dication.Similarly to the above pattern protonation of free bases 14a,b to form monocations leads to minor changes in nitrogen hfc, ΔaN = 0.02 mT, however more significant changes were observed upon the The EPR spectra of 3,4-disubstituted pyrrolidine nitroxides correspond to the pattern described for 1, with broad lines and two large additional doublet splittings on two hydrogen atoms of the methylene groups of the ethyl substituents [26,41] (Figure 5a).The value of these constants depends on the size of the neighboring substituent at position 3 or 4. For example, the introduction of the bulky maleimido group results in up to a 50% increase in one of two a H (cf. 14a and 15a; 16a and 17a).We have showed earlier that high hfc with one of methylene hydrogens of ethyl group is observed in nitroxides with the pseudoaxial position of this ethyl group and CH 2 -CH 3 bond nearly parallel to the N-O bond axis [41].Apparently, the repulsion of the large substituent and the neighboring ethyl group (cis relative to each other) increases the contribution of this conformation.In contrast, EPR spectra of the pyrroline nitroxides showed classical triplet with no additional large splittings (Figure 5b).In oxygen-free solution, hyperfine structure of the spectral lines was resolved, which is typical for the nitroxides with planar heterocycle (pyrroline, 3-imidazoline and isoindoline) (Figure 5d) [4].The simulation gives four a H = 0.0611 mT and four a H = 0.0317 mT, apparently due to hfc with methylene hydrogens of ethyl groups.
The EPR spectra of nitroxides with ionizable groups may be dependent on pH.Similarly to that described for 3-aminomethyl-proxyl (25) [42] (Figure 4), spectra of 3aminomethyl-and 3-dimethylaminomethyl nitroxides 6, 11a,b, 13a,b and 15a,b demonstrate small dependence on pH with ∆a N ca.0.02 mT and pK ca. 9. Titration of the nitroxides 14a and 14b revealed two transitions at pH ca. 3 and ca.11 (Figure 6), which apparently correspond to two protonation steps with formation of mono-and dication.Similarly to the above pattern protonation of free bases 14a,b to form monocations leads to minor changes in nitrogen hfc, ∆a N = 0.02 mT, however more significant changes were observed upon the second protonation, ∆a N = 0.044-0.055mT.Surprisingly, the constants on the hydrogen atoms of the methylene fragments of ethyl groups in 14a turned out to be more sensitive to pH changes (Figure 6).While formation of monocation 14aH + leads to decrease of apparent a H by 0.02 mT, the spectrum of 14aH 2 ++ (pH = 1) shows increase of the hydrogen hfc constant by 0.075 mT.Presumably the monocations 14aH + and 14bH + are stabilized due to coordination (hydrogen bonding) with the neighboring amino group forming 7-membered rings (Scheme 8).
second protonation, ΔaN = 0.044-0.055mT.Surprisingly, the constants on the hydrogen atoms of the methylene fragments of ethyl groups in 14a turned out to be more sensitive to pH changes (Figure 6).While formation of monocation 14aH + leads to decrease of apparent aH by 0.02 mT, the spectrum of 14aH2 ++ (pH = 1) shows increase of the hydrogen hfc constant by 0.075 mT.Presumably the monocations 14aH + and 14bH + are stabilized due to coordination (hydrogen bonding) with the neighboring amino group forming 7-membered rings (Scheme 8).This stabilization partly accounts for higher pK of the first protonation of 14a,b.Formation of these cyclic structures apparently decreases the repulsion of the ethyl groups and the substituents in the position 3 and 4. A noticeable decrease of aH upon the first protonation of 14a confirms the assumption of the formation of these cyclic structures.In contrast, the second protonation leads to opening of the 7-membered ring and strong conformational changes because of electrostatic repulsion of the neighboring cationic groups.These changes result in pronounced growth of aH in the spectra of 14a.In the spectra of 14b, the hyperfine structure of the triplet manifolds was not resolved, but growth of aH upon the second protonation can be followed by the inhomogeneous line broadening which leads to notable decrease of the peak height in the spectrum (Figure 6c).second protonation, ΔaN = 0.044-0.055mT.Surprisingly, the constants on the hydrogen atoms of the methylene fragments of ethyl groups in 14a turned out to be more sensitive to pH changes (Figure 6).While formation of monocation 14aH + leads to decrease of apparent aH by 0.02 mT, the spectrum of 14aH2 ++ (pH = 1) shows increase of the hydrogen hfc constant by 0.075 mT.Presumably the monocations 14aH + and 14bH + are stabilized due to coordination (hydrogen bonding) with the neighboring amino group forming 7-membered rings (Scheme 8).This stabilization partly accounts for higher pK of the first protonation of 14a,b.Formation of these cyclic structures apparently decreases the repulsion of the ethyl groups and the substituents in the position 3 and 4. A noticeable decrease of aH upon the first protonation of 14a confirms the assumption of the formation of these cyclic structures.In contrast, the second protonation leads to opening of the 7-membered ring and strong conformational changes because of electrostatic repulsion of the neighboring cationic groups.These changes result in pronounced growth of aH in the spectra of 14a.In the spectra of 14b, the hyperfine structure of the triplet manifolds was not resolved, but growth of aH upon the second protonation can be followed by the inhomogeneous line broadening which leads to notable decrease of the peak height in the spectrum (Figure 6c).This stabilization partly accounts for higher pK of the first protonation of 14a,b.Formation of these cyclic structures apparently decreases the repulsion of the ethyl groups and the substituents in the position 3 and 4. A noticeable decrease of a H upon the first protonation of 14a confirms the assumption of the formation of these cyclic structures.In contrast, the second protonation leads to opening of the 7-membered ring and strong conformational changes because of electrostatic repulsion of the neighboring cationic groups.These changes result in pronounced growth of a H in the spectra of 14a.In the spectra of 14b, the hyperfine structure of the triplet manifolds was not resolved, but growth of a H upon the second protonation can be followed by the inhomogeneous line broadening which leads to notable decrease of the peak height in the spectrum (Figure 6c).

Lipophilicity
Partition in octanol-water mixtures for ionizable compounds may depend on pH, and the pH changes may result from addition of the compound.Therefore, phosphate-citrateborate buffer (5 mM, pH 7.4) was used for K p measurements to make the partition closer to physiological conditions.The data is listed in Table 1.The new set of spin labels contains highly hydrophilic nitroxides, such as the positively charged spin labels 16a,b and 17a,b and negatively charged 20, which were not detected in octanol after shaking with buffer.Although monoamino derivatives 11 and 13 mainly exist in protonated form at pH 7.4, they still remain lipophilic and, therefore, should permeate cells.Amino groups are known to provide higher retention/intracellular accumulation due to transmembrane potential or ion-trapping effect [44].This effect was exploited in design of nitroxide contrast agents for MRI and radioprotectors [45].Cellular accumulation of spin labels like 11 and 13 may help to increase efficacy spin labeling using CuAAC or SPAAC.

Reduction Rates in Ascorbate-Containing Systems
Rate constants of the nitroxides decay in the ascorbate-containing systems are given in Table 1.All the pyrrolidine radicals demonstrated very high stability to reduction, exceeding that of 21.Reduction rates of pyrroline nitroxides are always expectedly higher than those of similar nitroxides of pyrrolidine series, but they were never higher than that of 21 by more than 40%.

Decay in the Rat Liver Homogenates
For this research we used a selection of nitroxides, containing cationic groups 17a,b, ionizable groups existing at pH 7.4 in cationic 14a,b and anionic 20, 21, 23 forms, and nonionizable nitroxides with hydroxyl groups 1, 22.We avoided using maleimido derivatives because they could react with proteins of the homogenate, therefore, the results would be hard to interpret.The nitroxide solutions were mixed with rat liver homogenate at 37 • C and decay of the EPR signal was followed.The kinetics of decay are shown in Figure 7.

Lipophilicity
Partition in octanol-water mixtures for ionizable compounds may depend on pH, and the pH changes may result from addition of the compound.Therefore, phosphate-citrateborate buffer (5 mM, pH 7.4) was used for Kp measurements to make the partition closer to physiological conditions.The data is listed in Table 1.The new set of spin labels contains highly hydrophilic nitroxides, such as the positively charged spin labels 16a,b and 17a,b and negatively charged 20, which were not detected in octanol after shaking with buffer.Although monoamino derivatives 11 and 13 mainly exist in protonated form at pH 7.4, they still remain lipophilic and, therefore, should permeate cells.Amino groups are known to provide higher retention/intracellular accumulation due to transmembrane potential or ion-trapping effect [44].This effect was exploited in design of nitroxide contrast agents for MRI and radioprotectors [45].Cellular accumulation of spin labels like 11 and 13 may help to increase efficacy spin labeling using CuAAC or SPAAC.

Reduction Rates in Ascorbate-Containing Systems
Rate constants of the nitroxides decay in the ascorbate-containing systems are given in Table 1.All the pyrrolidine radicals demonstrated very high stability to reduction, exceeding that of 21.Reduction rates of pyrroline nitroxides are always expectedly higher than those of similar nitroxides of pyrrolidine series, but they were never higher than that of 21 by more than 40%.

Decay in the Rat Liver Homogenates
For this research we used a selection of nitroxides, containing cationic groups 17a,b, ionizable groups existing at pH 7.4 in cationic 14a,b and anionic 20, 21, 23 forms, and nonionizable nitroxides with hydroxyl groups 1, 22.We avoided using maleimido derivatives because they could react with proteins of the homogenate, therefore, the results would be hard to interpret.The nitroxide solutions were mixed with rat liver homogenate at 37 °C and decay of the EPR signal was followed.The kinetics of decay are shown in Figure 7. Surprisingly, the decay rates showed no correlation with the reduction rates with ascorbate, with the lipophilicity values or with the nitroxide heterocyclic ring type.The Surprisingly, the decay rates showed no correlation with the reduction rates with ascorbate, with the lipophilicity values or with the nitroxide heterocyclic ring type.The compounds can be divided into three categories in accordance with the type of the functional groups.The decay of cationic nitroxides and amino derivatives was significantly faster than that of carboxylic acids and diols.In our previous study, we showed that the decay of positively charged 2,2,5,5-tetraethylpyrrolidine-1-oxyls in homogenates of kidney and liver proceeds much faster than in blood and in homogenates of brain and heart [22].
Therefore, a relatively high rate of decay of cationic nitroxides in liver homogenates does not mean that they cannot be used as spin labels in living cells, since the cells normally used in such studies do not have such a high activity towards xenobiotics.However, the above data show that negatively charged or non-charged polar nitroxides may perform better in EPR/NMR imaging studies in vivo.This coincides with the conclusions of the recent paper by Peyrot, et al. [24].Despite much similarity of our results to literature data, we are far from the assignment of the observed decay of nitroxides to certain enzyme activity and from elucidation of the decay mechanism without special investigation.

General Information
The compounds 1 [26,27] and 2 [28] were prepared according to the literature.The IR spectra were recorded on a Bruker Vector 22 FT-IR spectrometer (Bruker, Billerica, MA, USA) in KBr pellets (1:150 ratio) or in neat samples (see the Supplementary Materials in this article pp.S10-S18) and are reported in wave numbers (cm −1 ).UV spectra were acquired on a HP Agilent 8453 spectrometer (Agilent Technologies, Santa Clara, CA, USA) in ethanol solutions (concentration~10 −4 M). 1 H NMR spectra were recorded on a Bruker AV 300 (300.132MHz), AV 400 (400.134MHz) and DRX 500 (500.130MHz) spectrometers (Bruker, Billerica, MA, USA). 13C NMR spectrum was recorded on a Bruker AV 300 (75.467MHz) (see the Supplementary Materials in this article pp.S4-S9).All the NMR spectra were acquired for 5-10% solutions in CDCl 3 or CD 3 OD at 300 K using the signal of the solvent as a standard.To confirm the structure of stable nitroxides, 1 H NMR spectra were recorded of the solutions of corresponding hydroxylamines prepared via reduction of the nitroxide samples (10-20 mg) with Zn powder in a CD 3 OD-CF 3 COOH 10:1 mixture [22].HRMS analyses were performed using a High-Resolution Mass Spectrometer DFS (Thermo Electron, Waltham, MA, USA).

A Reaction of Hydroxymethyl Nitroxides 1 and 8 with Methanesulfonyl Chloride
Method A: N,N-diisopropylethylamine (DIPEA) was added dropwise within 6 h to a stirred solution of 1 and methanesulfonyl chloride 1.4 g in dry chloroform, the amount and ratio of the reagents is shown in Table 2.The mixture was stirred overnight, washed with water and dried with MgSO 4 .The solution was concentrated in a vacuum and separated using column chromatography on silica gel, eluent chloroform; yields of 3 and 4 are shown in the Table 2. Method B: Methanesulfonyl chloride (227 mg, 1.98 mmol) was added dropwise to a stirred solution of 8 (274 mg, 0.57 mmol) and DIPEA (335 mg, 2.59 mmol)) in dry chloroform (10 mL).The mixture was stirred at ambient temperature for 3 days (TLC control on silica gel, chloroform-methanol 100:1, visualization with UV-254), washed with water and dried with Na 2 CO 3 .The solution was concentrated in vacuum.The residue was purified using column chromatography on silica gel, eluent chloroform-methanol 100:1 to give 9 (247mg, 74%).

Reaction of Mesylates with Sodium Azide (General Method)
A mixture of a mesylate 3, 4, or 9 (12 mmol), NaN 3 (5 g, 77 mmol) and DMSO (50 mL) was heated to 60 • C and stirred for 4-6 h.The conversion was controlled using TLC (silica gel, chloroform, UV 254).The mixture was diluted with H 2 O (200 mL) and extracted with hexane-diethyl ether mixture 1:1 (3 × 100 mL).The extract was washed with water and dried with MgSO 4 .The solvent was distilled off and residue was purified as described below.

General Procedure of the Synthesis of Maleimides 7 and 15a,b
A solution of N-methoxycarbonyl maleimide NMCM (51 mg, 0.33 mmol) in absolute THF (10 mL) was cooled in an ice bath and the solution of nitroxide 6 or 14a,b (0.30 mmol) in absolute THF (5 mL) was added dropwise.The reaction mixture was allowed to stay under stirring for 5 min.The conversion was controlled using TLC (silica gel, methanol-ethyl acetate 1:9, UV 254).The mixture was diluted with saturated aqueous solution of NaHCO 3 (10 mL) and allowed to stay under stirring for 1 h.The conversion was controlled using TLC (silica gel, hexane-ethyl acetate 1:1, UV 254).The mixture was diluted with water and extracted with ethyl acetate (3 × 10 mL).The extract was washed with brine and dried with Na 2 SO 4 .The solvent was distilled off and residue was purified by column chromatography on silica gel (eluent hexane-ethyl acetate 1:1).

EPR Measurements and Kinetics
The stock solutions of the nitroxides in DMSO (20 mM) were diluted 100-fold with 5 mM phosphate-citrate-borate buffer pH 7.4.EPR spectra were recorded on Elexsys E540 X-band spectrometer (Bruker Corporation, Billerica, MA, USA) in a 50 µL glass capillary for 0.2 mM solutions, with the following spectrometer settings: frequency, 9.87 GHz; centerfield, 350.6 mT; sweep range, 10 mT; microwave power, 2.0 mW; modulation amplitude, 0.05 mT; time constant, 10.24 ms; and conversion time, 20.48 ms.Simulations of solution electron spin resonance lines were carried out in the EasySpin software (5.2.35), which is available at http://www.easypin.org(accessed on 18 May 2023).The spectra with resolved hyperfine structure were obtained for degassed solutions using the following settings: microwave power, 2.0 mW; modulation amplitude, 0.005 and 0.0025 mT; modulation frequency 100 KHz.
The pH-dependences of the EPR spectra were investigated using the 0.2 mM solutions of the nitroxides in 5 mM phosphate-citrate-borate buffer on a Bruker ER-200D-SRC spectrometer in 50 µL glass capillary for 0.2 mM radical solutions.Spectrometer settings: frequency, 9.87 GHz; microwave power, 5.0 mW; modulation amplitude, 0.1 mT; time constant, 50-100 ms; and conversion time, 50.12 ms.Small portions of 0.1-1 N HCl or 0.1-1 N NaOH were added to the stirred solution of a nitroxide in buffer at 25 • C, while the pH was controlled using pH-meter.After the equilibrium was established, the samples were taken from the solution and the EPR spectra were measured.The experimental points were fitted with the Equation (1) [43] to obtain the pKa values.
a N (pH) = (p 1 + p 2 × 10 pK 1 −pH + p 3 × 10 pK 1 +pK 2 −2×pH ) 1 + 10 pK 1 −pH + 10 pK 1 +pK 2 −2×pH (1) For kinetic measurements in water, stock solutions of nitroxide, ascorbic acid and glutathione in phosphate-citrate-borate buffer (5 mM, pH 7.4) were prepared, and pH was adjusted to 7.4 with NaOH or HCl.All the solutions were deoxygenated with argon, carefully and quickly mixed in a small tube to attain final concentrations (nitroxide, 0.2-04 mM; GSH, 5 mM; and ascorbate, 100-300 mM) and were placed into an EPR capillary (50 µL).The choice of acsorbate concentration is a compromise between the low reduction rate, which requires very long experiment time and deviations of the kinetics from linearity at higher ascorbate concentrations due to ionic strength effects.Since the ascorbate oxidation is slow under these conditions, the 5 mM concentration of GSH was sufficient to suppress the reverse reaction of ascorbate radical with the hydroxylamine, because the rate of the ascorbate radical formation was low.The increase in GSH concentration produced little effect on the final reduction rate constant values.The capillary was sealed on both sides and placed into the EPR resonator.The decay of amplitude of the low-field component of the EPR spectrum was followed to obtain the kinetics.The initial part of the decay curves (up to 20 min) was used for fitting.Kinetics of the decay was fitted to a monoexponential function to calculate the first-order rate constants.Then, these constants were divided by the concentration of ascorbic acid to calculate the second-order reaction constants.Partition coefficients in a water-octanol mixture were estimated from the amplitudes of the EPR spectra of a nitroxide in water after extensive shaking with different portions of added octanol and separation using centrifugation.
The measurements of kinetics of nitroxide decay in liver homogenates were performed using Bruker ER-200D-SRC spectrometer in a 50 µL glass capillary for 0.2 mM solutions, with the following spectrometer settings: frequency, 9.87 GHz; centerfield, 350.6 mT; sweep range, 10 mT; microwave power, 2.0 mW; modulation amplitude, 0.1 mT; time constant, 10.24 ms; and conversion time, 20.48 ms.Before measurement, 10 mM solutions of the nitroxides in PBS were prepared ex tempore and mixed with thawed homogenates (final concentration 0.2 mM).Homogenates were then loaded into 50 µL glass capillaries, which were then sealed with plasticine and inserted into the chamber of the EPR spectrometer.The time elapsed between thawing and beginning of the recording was 7 min.The decay of low-field component of the EPR spectrum was followed to obtain the kinetics.

Animals
Male Wistar rats weighing 220-250 g were kept under standard conditions with unlimited access to water and pelleted food.All manipulations with animals were carried out in a strict accordance with the legislation of the Russian Federation, GOST 33044-2014 "Principles of Good Laboratory Practice", GOST 33647-2015 "Principles of Good Laboratory Practice (GLP).Terms and definitions", Decision "On approval of the Rules of Good Laboratory Practice of the Eurasian Economic Union in the field of circulation of medicines" dated 3 November 2016 No. 81 and the provisions of Directive 2010/63/EU of the EU Parliament and the Council of the European Union dated 22 September 2010 on the protection of animals used for scientific purposes.The animal study protocol was approved by the Ethics Committee of the Institute of Organic Chemistry N.N.Vorozhtsov SB RAS (protocol No. P-05-06.2022-14,approved 5 June 2022).
Animals were sacrificed by decapitation, organ tissue (liver) was excised on ice, rinsed with ice-cold 10 mM PBS (pH 7.4), sheared with scissors and homogenized in PBS (1/1 by weight) on ice using electric homogenizer fitted with a Teflon head.The homogenates were snap-frozen in liquid nitrogen.

Conclusions
In this work, we tested various methods of synthesis heterofunctional derivatives of the sterically shielded nitroxide 1.The resulting maleimide and azide spin labels of both pyrrolidine and pyrroline series demonstrated high resistance to reduction.Besides that, they contain hydrophilic moieties, making them soluble in water and allowing for additional coordination with target biomolecules for more precise distance measurements.The new pyrroline spin labels are especially valuable due to simple EPR spectra with relatively narrow lines.
The decay of the new nitroxides in rat liver homogenates was studied.The results revealed higher stability of negatively charged and neutral nitroxides as compared to the positively charged ones.This information will be used in design of the spin probes for in vivo EPRI and MRI applications.
Another important finding in this work is the unexpectedly high sensitivity of the EPR spectra of 14a to pH changes.It is not only the case that the nitrogen hfc is decreasing by 0.055 mT upon second protonation (pK a 3.0), but also hydrogen hfc constants are increasing by 0.075 mT.Unless these effects are somewhat lower than pH-sensitivity of widely used spin probes [4] and pK is far from physiologically important range, this is the first example of a functional spin probe where variation of hfc on γ-hydrogens due to conformational changes is used.We believe that similar structures can be designed where comparable changes can result, for example, from a reaction with thiols (see [46]).High stability to reduction makes further investigation of these nitroxides very promising.

Scheme 2 .
Scheme 2. The reaction of 1 with MsCl in the presence of DIPEA.

Figure 1 .
Figure 1.The structure of 3, 4 and 6 according to single-crystal X-ray diffraction data (hydrogen atoms are not shown).

Scheme 2 .
Scheme 2. The reaction of 1 with MsCl in the presence of DIPEA.

Scheme 2 .
Scheme 2. The reaction of 1 with MsCl in the presence of DIPEA.

Figure 1 .
Figure 1.The structure of 3, 4 and 6 according to single-crystal X-ray diffraction data (hyd atoms are not shown).

Figure 1 .
Figure 1.The structure of 3, 4 and 6 according to single-crystal X-ray diffraction data (hydrogen atoms are not shown).

Scheme 4 .
Scheme 4. Conversion of 6 into spin label 7 and synthesis of 11a.

Scheme 4 .
Scheme 4. Conversion of 6 into spin label 7 and synthesis of 11a.

Scheme 4 .
Scheme 4. Conversion of 6 into spin label 7 and synthesis of 11a.

Figure 2 .
Figure 2. The structure of 8, 10 and 12a according to single-crystal X-ray diffraction data (hydrogen atoms are not shown).
The subsequent Staudinger reaction with one equivalent of triphenylphosphine afforded 13a,b in 64-73% yield.The resulting aminoazides were converted to 11a,b by Eschweiler-Clarke alkylation.

Figure 2 .
Figure 2. The structure of 8, 10 and 12a according to single-crystal X-ray diffraction data (hydrogen atoms are not shown).
The addition of methyl iodide to the solutions of the spin labels 11a,b and 15a,b in dry diethyl ether leads to precipitation of the corresponding trimethylammonium iodides 16a,b and 17a,b.

Figure 3 .
Figure 3.The structure of 15a,b and 20 according to single-crystal X-ray diffraction data (hydrogen atoms are not shown).

Figure 3 .
Figure 3.The structure of 15a,b and 20 according to single-crystal X-ray diffraction data (hydrogen atoms are not shown).

Figure 6 .
Figure 6.Dependence of the EPR spectra of 14a,b on pH.Titration curves, 14a, a N versus pH (blue squares), a H versus pH (red circles), 14b, a N versus pH (green diamonds) (a).The fitting with modified Henderson-Hasselbalch equation [43] is shown with solid lines.EPR spectra of 14a at pH 1.0, 7.0 and 13.4 (b); of 14b at pH 1.7 and 6.8 (c).

Table 1 .
• C in the presence of glutathione 5 mM.For spectrometer settings see Section 3. Parameters of the EPR spectra of nitroxides in 50 mM phosphate buffer (pH 7.4) with the addition of 1% DMSO at concentration of 0.2 mM (aN-hfc constant on nitrogen, aH-hfc constant on methylene hydrogens of ethyl groups, Hp-p-peak to peak linewidth), partition coefficients (Kp) in octanol/aqueous 5 mM phosphate-citrate-borate buffer pH 7.4 mixtures, and rates of reduction with ascorbate (kred) (100-300 mM) in 5 mM phosphate-citrate-borate buffer, pH 7.4, 25 °C in the presence of glutathione 5 mM.For spectrometer settings see Materials and Methods section.